The present invention relates to a dry extract which is rich in isoflavones, as aglycones, and to a process for preparing the same.
More particularly, the present invention relates to a dry extract containing at least 8% (w/w) of one or more isoflavones, as aglycones, of formula 
wherein
R1=OH and R2=H, Genistein;
R1=H and R2=H, Daidzein;
R1=H and R2=CH3, Formononetin; and
R1=OH and R2=CH3, Biochanin A.
It is known that isoflavones belong to the class of phytoestrogens or plant oestrogens. These are particularly useful thanks to their pharmacological and therapeutic properties such as, for example, their anti-cancer and oestrogen activity (WO 99/48496, WO 99/43335 and WO 93/23069).
Such properties have been mainly attributed to the aglycone form of the isoflavones, i.e. to the isoflavone moiety without the sugar moiety.
In nature, isoflavones are present in many plant materials such as, for example, Trifolium pratense, Trifolium subterranean, Abrus precatorius, various species of Acacia, Apio tuberosa, Arachis hypogea, Astragalus edulis, Bauhinina esculenta, Cajanus cajan indicus, Canavalia ensiformis, Canavalia gladiata, Canavalia rosea, varies species of Cassia, Ceratonia siliqua, Cicer arietinum, Cordeauxia edulis, various species of Crotalaria, Cyramopsis psoralioides, Detariaum senegalese, Entada scandes, Erythrina edulis, Glycine max, Ingaedulis, Inocarpus fagifer, Lablab purpureus, Lathyrus sativus, Lathyrus ochrus, Lens culinaris, Leucaenal eucocephala, various species of Lupinus, Macatylma geacapra, Macrotyloma uniflorum, Medicago sativa, Mucuna pruriens, Pachyrhyzuz erosus, Parkia clappertoniana, Parkia speciosa, Pentaclethra macrophylla, various species of Phaseolus, Pisum sativum, Pithecolobium lobatum, various species of Prosopis, Psophocarpus scandens, various species of Psoralea, Sesbania bispinosa, Sphenostylis stenocarpa, Tamarindus indica, Trigonella foenum-graecum, various species of Vivia, Vicia faba, Vigna mungo, various speices of Vigna and Voandzeia subterranea. 
Patent application WO 93/23069 describes compositions that are rich in natural phytoestrogens or analogues thereof, such as Genistein, Daidzein, Formononetin and Biochanin A.
These phytoestrogens are obtained by:
a) drying the plant material;
b) extracting it with a mixture of water and a water-miscible organic solvent (60% ethanol, Example 1);
c) separating the extract from the undissolved plant material and removing the organic solvent; and
d) concentrating the aqueous phase.
However, in the said document, the extraction yield is never indicated and it is not specified whether the isoflavones are obtained as aglycones or in glycosylated form.
Patent application WO 99/43335 describes compositions of an extract of clover from which have been isolated compounds containing aromatic chromophoric groups such as the isoflavones Genistein, Daidzein, Formononetin and Biochanin and/or glucosides thereof. The said extract has anticancer activity against one or more HL60, K562, LNCaP or HT29 cell lines. The said composition is optionally in combination with a pharmaceutically acceptable carrier, auxiliary excipients and/or diluents.
This extract is prepared by:
a) drying the plant material;
b) extracting the thus obtained material with a mixture of water and a water-miscible organic solvent (60% ethanol, Example 1);
c) separating the extract from the undissolved plant material and removing the organic solvent;
d) extracting the aqueous phase with an organic solvent such as petroleum ether;
e) removing the organic solvent;
f) drying the aqueous phase; and
g) separating the isoflavones containing aromatic chromophoric groups by HPLC (page 6, lines 10-29).
This document too never indicates the extraction yield. In addition, even the experimental section fails to indicate whether the isoflavones are obtained as aglycones or in glycosylated form.
International patent application WO 99/48496 describes a method for the treatment, prophylaxis, improvement or prevention of conditions associated with an abnormally high activity of steroidal oestrogens, the said method comprising the administration of a composition of an extract containing clover or chick pea isoflavones, the said extract mainly comprising biochanin, or a biochanin/formononetin, daidzein and genistein ratio of between 2:1 and 5:1 approximately, optionally in combination with a pharmaceutically acceptable carrier, auxiliary excipients and/or diluents.
This extract is prepared by:
a) freezing the fresh plant material with liquid nitrogen;
b) grinding the thus obtained material;
c) extracting the thus obtained material with a mixture of water and a water-miscible organic solvent (60% ethanol, Example 1);
d) separating the extract from the undissolved plant material and removing the organic solvent;
e) extracting the aqueous phase with an organic solvent such as petroleum ether, hexane or ethyl acetate;
f) removing the organic solvent; and
g) drying the aqueous phase (page 8, lines 4-32).
However, the extraction yield is never indicated in this document. In addition, in the experimental section it is reported that the aqueous phase contains the isoflavones as aglycones (Example 1, page 12, line 26). However, this must be an entirely negligible amount since it is known that the aglycones are substantially insoluble in water.
There is therefore still a great need for a dry extract containing at least 8% (w/w) of one or more isoflavones, as aglycones, and for a process for preparing the same.
It has now been surprisingly found that this result is achieved if, before extracting the isoflavones with an organic solvent, the dry plant material undergoes a maceration in water, preferably under an inert atmosphere and in a vessel protected against light. Indeed, it has been found that the isoflavone moiety separates from the sugar moiety in said maceration step. The reason for this has still not been fully understood but, without hereby limiting the present invention, it is opined that the isoflavone-sugar link is labile enough to be hydrolysed in water and/or that the plant material contains an enzyme capable of hydrolysing the abovementioned link.
It is, therefore, a first object of the present invention to provide a process for extracting isoflavones from a plant material, wherein the said process comprises treating the plant material with an organic solvent, characterized in that the said extraction is preceded by a maceration step in water.
The said organic solvent is preferably miscible with water.
Typical examples of the said water-miscible organic solvents are alkanols having from 1 to 4 carbon atoms such as, for example, methanol, ethanol, propanol and butanol. Other typical examples of the said solvents are propylene glycol, erythritol, butanediol, acetonitrile, ethylene glycol, ethyl acetate, glycidol, dihydroxyacetoneglycerol and acetone.
Advantageously, the plant material, which undergoes maceration, is in dry form. Even more advantageously, the plant material is also ground up.
The said maceration is preferably carried out under an atmosphere of an inert gas such as nitrogen.
According to a preferred embodiment, the said maceration is carried out in a vessel protected against light.
The said maceration is advantageously carried out at a temperature of from 20xc2x0 C. to 30xc2x0 C.
The maceration time may vary within a wide range depending on parameters that are well known to those skilled in the art, such as, for example, the temperature and volume of water present per unit by weight of dry plant material. As a guide, this time is of from 10 minutes to 24 hours. This time is preferably of from 2 to 6 hours.
The abovementioned maceration is typically followed by the following steps:
a) extraction with at least one water-miscible organic solvent,
b) separation of the undissolved plant material,
c) treatment of the solution obtained in step b) with an aliphatic hydrocarbon having from 5 to 8 carbon atoms, and
d) removal of the solvent in order to obtain a solid residue.
The thus obtained solid residue contains at least 8% (w/w) of one or more isoflavones in the form of aglycones.
The treatment in the abovementioned step c) is mainly intended to remove the essential oils such as, for example, terpenes, and waxes.
The aliphatic hydrocarbon in the abovementioned step c) is preferably n-heptane.
After step d), the following steps may optionally also be carried out:
e) optionally, redissolution of the solid residue from step d) in a water-miscible organic solvent,
f) extraction of the solution obtained in step e) with a suitable organic solvent, and
g) removal of the solvent in order to obtain a solid residue.
Suitable solvents which may be used in the abovementioned step f) are those which have high affinity for isoflavones in the form of aglycones, such as, for example, methyl tert-butyl ether and ethyl acetate.
It is, therefore, a further object of the present invention to provide a dry extract containing, as aglycones, at least 8% (w/w) of one or more isoflavones of formula 
wherein
R1=OH and R2=H, Genistein;
R1=H and R2=H, Daidzein;
R1=H and R2=CH3, Formononetin;
R1=OH and R2=CH3, Biochanin A.
The amount of the abovementioned aglycones is preferably greater than 15% (w/w).
The examples which follow are given to illustrate the present invention without, however, limiting it in any way.